Mosher's amide-based assignment of the absolute configuration of phantasmidine

Article abstract of DOI:10.3987/COM-13-S(S)45

The absolute configuration of phantasmidine was assigned by analysis of the two conformers of the two Mosher amides. The expected shielding of the protons adjacent to the nitrogen and large long range shielding (～1 ppm) of the cyclobutane methylene protons in 16major and the pyridine protons of 16minor by the phenyl group of the Mosher amide allows the assignment of absolute configuration as shown in Schemes 4 and 5.

Full text of DOI:10.3987/COM-13-S(S)45

HETEROCYCLES, Vol. 88, No. 1, 2014
779
HETEROCYCLES, Vol. 88, No. 1, 2014, pp. 779 - 787. © 2014 The Japan Institute of Heterocyclic Chemistry
Received, 25th June, 2013, Accepted, 5th August, 2013, Published online, 7th August, 2013
DOI: 10.3987/COM-13-S(S)45
MOSHER'S AMIDE-BASED ASSIGNMENT OF THE ABSOLUTE
CONFIGURATION OF PHANTASMIDINE
Quan Zhou and Barry B. Snider*
Department of Chemistry MS 015, Brandeis University, 415 South Street, Waltham,
Massachusetts 02454-9110, USA; E-mail: snider@brandeis.edu
Abstract – The absolute configuration of phantasmidine was assigned by analysis
of the two conformers of the two Mosher amides. The expected shielding of the
protons adjacent to the nitrogen and large long range shielding (~1 ppm) of the
cyclobutane methylene protons in 16major and the pyridine protons of 16minor by
the phenyl group of the Mosher amide allows the assignment of absolute
configuration as shown in Schemes 4 and 5.
Dedicated to Professor Victor Snieckus on the occasion of his 77th birthday
In 1992 Daly isolated the much studied epibatidine (8) from the skin of the Ecuadorian poison frog
Epipedobates anthonyi.1 In 2010 Fitch, Daly and co-workers isolated the minor, rigid, tetracyclic congener
phantasmidine (7) from the same frog.2 The structure of phantasmidine was tentatively assigned
spectroscopically from HPLC-purified 7 and the derived acetamide 17 with a total sample size of only 20
μg. We recently reported a short and efficient synthesis of racemic phantasmidine (7) that confirmed the
structure assignment.3 Deprotonation of 2-fluoro-6-chloropyridine (1) at the 3-position with LDA and
trapping of the resulting carbanion with DMF provided 2, which was elaborated to primary amide 3.
Coupling of primary amide 3 with protected acyloin 4 gave racemic secondary amide 5, which was treated
with potassium hydroxide in t-butanol to effect an intramolecular aldol reaction and nucleophilic aromatic
displacement to afford tetracyclic pyrrolidinone 6, which was reduced with borane to give racemic
phantasmidine (7) in 8 steps and 8% overall yield (see Scheme 1).
The coupling of amide 3 with 4 gave racemic 5, making this route unsuitable for the synthesis of
enantiomerically pure phantasmidine. Fortunately, the two enantiomers were very readily separated by
chiral HPLC on a Chiralcel OJ-H column with retention times of 29 and 44 minutes, making both
enantiomers readily available for biological investigation.3 The optical rotation of the faster eluting
enantiomer (+)-phantasmidine is []25 +70 (c 0.17, CH2Cl2) and that of the slower eluting enantiomer
D

780
HETEROCYCLES, Vol. 88, No. 1, 2014
(-)-phantasmidine is []25D -77 (c 0.17, CH2Cl2). The two enantiomers of epibatidine (8) have very similar
shapes and very similar biological activity.1 We expected that there would be a significant difference in the
biological activity of the two enantiomers of the much more rigid tetracyclic congener phantasmidine
making it important to assign the absolute configuration, which we report here.
Scheme 1. Synthesis of (±)-phantasmidine (7)
Although (±)-phantasmidine is highly crystalline, we were unable to obtain suitable crystals of the pure
enantiomers obtained by chiral HPLC. Initial attempts at resolving larger quantities and forming a
crystalline salt suitable for X-ray crystallography by addition of 0.5 equivalent of various enantiomerically
pure acids to racemic phantasmidine were also unsuccessful. We then turned our attention to the use of
Mosher's amides as developed by Hoye, who prepared the diastereomeric Mosher's amides 9 and 10 from
1
(2R,5R)-2,5-dimethylpyrrolidine and showed that the H NMR spectra are remarkably different as
indicated in Scheme 2.4-6 Conformational studies indicated that the predominant conformer has the
trifluoromethyl group syn to the carbonyl group as shown. The assignment was facilitated by the
C2-symmetry of (2R,5R)-2,5-dimethylpyrrolidine which results in only a single amide conformer. One
methyl group, presumably that adjacent to the carbonyl group absorbs at  1.30 and  1.32 in the two
diastereomers. As expected, the methyl group shielded by the phenyl group in 10 ( 0.10) is shifted upfield
by 0.99 ppm from the methyl group adjacent to the methoxy group in 9 ( 1.09). Similarly, the methine
Scheme 2. NMR absorptions for the Mosher's amides of (2R,5R)-2,5-dimethylpyrrolidine

HETEROCYCLES, Vol. 88, No. 1, 2014
781
hydrogen in 9 shielded by the phenyl group ( 3.19) is shifted upfield by 1.24 ppm from the methine
hydrogen adjacent to the methoxy group in 10 ( 4.43).
We had some concerns regarding the application of this procedure to phantasmidine.
(2R,5R)-2,5-Dimethylpyrrolidine is C2-symmetric so that there is only one amide conformer of 9 and 10.
The Mosher's amides of phantasmidine 15 and 16 will exist in two amide conformers on the NMR time
scale, as does N-acetylphantasmidine (17),2 making the assignment of absolute configuration more difficult.
MMX calculations by PCMODEL 8.07 indicated that the major amide conformers 11major and 12major
of the unsymmetrical (R)-2-methylpyrrolidine (R)-Mosher's amides 11 and (S)-Mosher's amide 12 with the
large Ph(OMe)CF3C group adjacent to the smaller methylene carbon are about 2 kcal/mol more stable than
the minor amide conformers 11minor and 12minor with the large Ph(OMe)CF3C group adjacent to the
large methyl-substituted methine carbon (see Scheme 3). As expected, only one conformer corresponding
1
to 11major and 12major was observed by H NMR spectroscopy for both diastereomers of the closely
related triethylsilyloxymethylpyrrolidine Mosher's amides 13 and 14.4c The methine protons of 13 and 14
on C-2 have almost identical chemical shifts ( = 0.02) whereas the methylene protons shifted
significantly  = 0.23 and -0.41). This suggested that in both cases the carbonyl group is adjacent to the
methyl-substituted methine proton whereas the major shielding factor, the phenyl group, is adjacent to the
methylene group. Other 2-alkylpyrrolidine Mosher's amide spectroscopic data support this analysis.5
Scheme 3. Conformational analysis and spectra of 2-alkylpyrrolidine Mosher's amides
To our surprise, MMX calculations by PCMODEL 8.0 suggested that the large Ph(OMe)CF3C group is
adjacent to the cyclobutane-substituted methine carbon in the major amide conformers (more stable by
0.9-1.2 kcal/mol) of phantasmidine Mosher's amides 15major and 16major rather than the methylene

782
HETEROCYCLES, Vol. 88, No. 1, 2014
carbon as in 11major and 12major (see Scheme 4). Similar calculations indicate that the methyl group of
phantasmidine acetamide (17) also prefers to be adjacent to the cyclobutane-substituted methine carbon by
1.1 kcal/mol. These calculations suggest that the methylene group of the cyclobutane fused to the
pyrrolidine is smaller than a proton, whereas the C-2 methyl group of a pyrrolidine is obviously much larger
than a proton. Apparently, the ~90° bond angles in the cyclobutane move the cyclobutane methylene carbon
away from the nitrogen so that it is effectively smaller than a proton.
Cl
Cl
6.93
6.91
7.37
H
N
H
N
(-)-phantasmidine
(S)-Mosher's Amide
3:1 observed
G = 0.65 kcal/mol (obs)
G = 1.2 kcal/mol (calc)
O
O
H
7.49
3.75
3.95
O
R
H
2.31, 2.64
1.40, 2.50
H
2.21, 2.39
3.67
3.04
3.91
Ph
R
H
S
N
H
1.70, 2.35
H
H
S
N
H
H
4.30
H
5.04
4.74
3.59
MeO
Ph
S
O
S
CF3
OMe 3.28
F3C
15major
15minor
Cl
Cl
6.91
H
6.56
6.41
(+)-phantasmidine
(S)-Mosher's Amide
4:1 observed
G = 0.82 kcal/mol (obs)
G = 0.9 kcal/mol (calc)
H
N
N
O
H
7.48
H
O
S
2.31, 2.66
1.63, 2.49
H
2.02, 2.23
3.63
3.78
3.88
Ph
S
H
3.80
4.41
3.98
R
N
H
0.68, 0.87
R
N
H
H
H
H
4.71
H
5.25
3.34
MeO
Ph
O
S
O
S
CF3
OMe 3.77
F3C
16major
16minor
Cl
Cl
N
N
N
phantasmidine acetamide
2:1 observed
O
O
4.15
4.08
G = 0.41 kcal/mol (obs)
G = 1.1 kcal/mol (calc)
H
H
4.32
3.96
4.02 (shielded ax)
H
H
H
H
N
H
4.79
H
4.70
4.08 (deshielded eq)
(deshielded)
O
17major
O
17minor
Scheme 4. Conformational analysis and spectra of phantasmidine Mosher's amides
Acylation of readily available synthetic (±)-phantasmidine (7) with (R)-Mosher's acid chloride (prepared
1
from the (S)-Mosher's acid)8 gave a mixture of diastereomers 15 and 16. The H NMR spectrum was

HETEROCYCLES, Vol. 88, No. 1, 2014
783
complex with two amide conformers for each diastereomer. We were able to assign the peaks for each of the
four amide conformers from the COSY spectrum of the mixture. Acylation of 0.5 mg of (-)-phantasmidine
and 0.5 mg of (+)-phantasmidine with R-Mosher's acid chloride8 in CDCl3 gave the corresponding
(-)-phantasmidine S-Mosher's amide 15 and (+)-phantasmidine S-Mosher's amide 16, respectively. The
crude products were washed with 5% aq. HCl and used for spectroscopic analysis without further
purification. The 1H NMR spectrum of the purified mixture of diastereomers 15 and 16 was used to exclude
impurities due to unreacted Mosher's acid or Mosher's acid chloride.
A 3:1 mixture of amide conformers was observed for (-)-phantasmidine S-Mosher's amide 15. The methine
hydrogen adjacent to the phenyl group in 15major ( 4.30) is shifted upfield by 0.74 ppm from the methine
hydrogen in 15minor ( 5.04). The methylene hydrogen adjacent to the phenyl group in 15minor ( 3.04)
is shifted upfield by 0.91 ppm from the methylene hydrogen in 15major (95). These shifts allow us to
assign the absolute configuration as shown and indicate that, as calculated, the methylene carbon is larger
than the cyclobutane-substituted methine carbon, although the observed G (0.65 kcal/mol) is smaller than
the calculated G (1.2 kcal/mol)).
A 4:1 mixture of amide conformers was observed for (+)-phantasmidine S-Mosher's amide 16. The
methylene hydrogens of the cyclobutane adjacent to the phenyl group in 16major ( 0.68,  0.87) are
shifted upfield by 0.95 and 1.62 ppm from those in 16minor ( 1.63,  2.49). The pyrrolidine methylene
proton and two aryl protons that are adjacent to the phenyl group in 16minor ( 3.78,  6.41, and  6.56)
are also shifted upfield significantly (0.63, 1.07, and 0.35 ppm) from those in 16major ( 4.41, 7.48, and
 6.91). These shifts allow us to assign the absolute configuration as shown and indicate that, as calculated,
the methylene carbon is larger than the cyclobutane-substituted methine carbon, although the observed G
(0.82 kcal/mol) is smaller than the calculated G (0.9 kcal/mol).
The exceptionally large  values established the absolute configuration of (-)-phantasmidine as shown in
15 and (+)-phantasmidine as shown in 16. The remarkably large shielding of the cyclobutane methylene
group of 16major and the pyridine protons of 16minor by the Mosher's amide phenyl group is consistent
with the MMX-minimized structures shown in Figure. 1. The close agreement of calculations and
experiment on the relative amide conformer energies provides further support for this assignment.
Phantasmidine acetamide (17) showed a 2:1 mixture of amide conformers in the 1H NMR spectrum whose
conformations were assigned from NOE data.2 The methine proton ( 4.79) adjacent to the carbonyl group
in the minor amide conformer 17minor is deshielded by the carbonyl group and shifted downfield 0.07
ppm from the methine proton adjacent to the methyl group in the major amide conformer 17major ( 4.70).
The methylene protons adjacent to the nitrogen were assigned based on the cis vicinal coupling constant
(7.0-8.4 Hz, 20-30°) being larger than the trans vicinal coupling constant (3.2 Hz, 95-110°). The

784
HETEROCYCLES, Vol. 88, No. 1, 2014
pseudoequatorial methylene hydrogen is in the deshielding cone of the carbonyl group and is shifted
downfield to  4.08 in the major amide conformer from  3.96 in the minor amide conformer. The
pseudoaxial methylene hydrogen is in the shielding cone of the carbonyl group and is shifted upfield to 
4.02 from  4.32 in the minor amide conformer. The 2:1 mixture of amide conformers indicates that
17major is more stable by 0.41 kcal/mol, which is somewhat smaller than the calculated value of
1.1 kcal/mol, but does again indicate that the methylene group is larger than the cyclobutane-substituted
methine carbon.
(a)
(b)
Figure 1. (a) MMX-minimized structure of 16major showing shielding (δ 0.68 and 0.87) of the
cyclobutane methylene group by the phenyl group. (b) MMX-minimized structure of 16minor showing
shielding (δ 6.41 and 6.56) of the pyridine hydrogens by the phenyl group.
Both enantiomers of epibatidine have comparable biological activity.1 The absolute configuration of
epibatidine was established as shown in 8 by chiral HPLC comparison with synthetic material of known
absolute configuration.9 The biosynthetic pathways to epibatidine and phantasmidine are not known nor is
it clear how they are biosynthetically related. The slower eluting (-)-phantasmidine ((-)-7) has the same
absolute configuration as natural epibatidine at the asterisked carbons (see Scheme 5). We are unable to
determine the absolute configuration of natural phantasmidine because a sample is no longer available.
Surprisingly, preliminary results indicate that faster eluting (+)-phantasmidine ((+)-7) is considerably more
biologically active than slower eluting (-)-phantasmidine.10 We believe that the assignment of the absolute
configuration of phantasmidine based on the Mosher's amides is secure because of the magnitude of the
chemical shift differences and we were carful to deal with the formal change that occurs when (S)-Mosher's
acid is converted to the (R)-Mosher's chloride and back to the (S)-Mosher's amide. This raises several
possibilities that can't be distinguished between at this time. Nature could make epibatidine and
(+)-phantasmidine with opposite absolute configuration at the asterisked carbons, nature could make the

HETEROCYCLES, Vol. 88, No. 1, 2014
785
less biologically active (-)- or racemic phantasmidine, or the absolute configuration of epibatidine could be
misassigned.
Scheme 5. Structures of natural epibatidine (8), less biologically active (-)-phantasmidine and more
biologically active (+)-phantasmidine
In conclusion, the absolute configuration of phantasmidine was assigned by MMX analysis of the two
1
conformations of Mosher amides 15 and 16 and their H NMR spectra. The expected shielding of the
protons adjacent to the nitrogen and large long range shielding (~1 ppm) of the cyclobutane methylene
protons in 16major and the pyridine protons of 16minor by the phenyl group of the Mosher amide allows
the assignment of absolute configuration as shown in Schemes 4 and 5.
EXPERIMENTAL
Racemic Phantasmidine (S)-Mosher's Amide (15 and 16). A mixture of (S)-Mosher's acid (8.5 mg,
36 mol) in 1 mL of hexanes was treated with 2 L of DMF and then 10 L (14.8 mg, 116 mol) of oxalyl
chloride at 25 °C. The reaction was kept at 25 °C for 30 min and filtered. The filtrate was concentrated to
give crude (R)-(-)-Mosher's acid chloride as colorless oil, which was used directly without further
purification.
A solution of racemic phantasmidine (7, 4 mg, 18 mol) and diisopropylethylamine (30 L, 170 mol) in
0.2 mL of anhydrous CH2Cl2 was treated with a solution of the crude (R)-(-)-Mosher's acid chloride in
0.5 mL of anhydrous CH2Cl2 at 25 °C under N2. The reaction was stirred at 25 °C for 1 h and quenched with
2 mL of 5% aqueous Na2CO3. The resulting mixture was extracted with CH2Cl2 (3 mL × 3) and the
combined organic extracts were dried (MgSO4) and concentrated. Flash chromatography of the residue on
silica gel (CH2Cl2 to 100:1 CH2Cl2/MeOH) gave 6.1 mg (77%) of a 1:1 mixture of (±)-phantasmidine
(S)-Mosher's amide diastereomers 15 and 16. One diastereomer exists as a 3:1 mixture of amide conformers
and the other as a 4:1 mixture.
(S)-Mosher's Amide of the Slower Eluting (-)-Phantasmidine Enantiomer (15). The identical
procedure using ~0.5 mg of (-)-phantasmidine gave a mixture of 15major and 15minor whose 1H NMR
spectrum showed a 3:1 mixture of amide conformers. The spectral data described below were obtained by
analysis of the spectrum of this crude diastereomer and the spectrum of the purified mixture of both

786
HETEROCYCLES, Vol. 88, No. 1, 2014
diastereomers described above.
Partial data not including the phenyl protons for 15major: 1H NMR  7.49 (dd, 1, J = 7.6, 1.2), 6.93 (d, 1, J
= 7.6), 4.74 (dd, 1, J = 12.8, 1.2), 4.30 (dd, 1, J = 8.4, 6.8), 3.95 (dd, 1, J = 12.8, 7.2), 3.75 (br d, 1, J = 7.2),
3.59 (q, 3, JH,F = 1.8, OMe), 2.42-2.32 (m, 2), 2.21 (br dd, 1, J = 12, 10.4), 1.74-1.66 (m, 1).
Partial data not including the phenyl protons for 15minor: 1H NMR  7.37 (presumed d, 1, assigned from
COSY spectrum), 6.91 (d, 1, J = 7.6), 5.04 (dd, 1, J = 7.6, 7.6), 3.91 (dd, 1, J = 12.8, 2,1), 3.67 (br d, 1, J =
7.6), 3.28 (q, 3, JH,F = 1.8, OMe), 3.04 (dd, 1, J = 12.8, 7.6), 2.64 (br ddd, 1, J = 12, 10, 10), 2.54-2.46 (m, 1),
2.35-2.27 (m, 1), 1.44-1.36 (m, 1).
(S)-Mosher's Amide of the Faster Eluting (+)-Phantasmidine Enantiomer (16). A solution of
(+)-phantasmidine (~0.5 mg, 2.2 mol) and dry pyridine (1 L, 12.5 mol) in anhydrous 100 L of CDCl3
was treated with (R)-(-)-Mosher's acid chloride (6.4 mol, prepared as described above from the
(S)-(-)-Mosher's Acid) in 100 L of CDCl3 at 25 °C under N2. The reaction was kept at 25 °C for 1 h and
diluted with 0.3 mL of dry CDCl3. The entire CDCl3 solution was transferred to an NMR tube for
1
spectroscopic analysis. The H NMR spectrum of this diastereomer showed a 4:1 mixture of Mosher's
amide conformers 16major and 16minor. The spectral data described below were obtained by analysis of
the spectrum of this crude diastereomer and the spectrum of the purified mixture of both diastereomers
described above.
Partial data not including the phenyl protons for 16major: 1H NMR  7.48 (dd, 1, J = 7.6, 1.2), 6.91 (d, 1, J
= 7.6), 4.71 (dd, 1, J = 7.6, 7.2), 4.41 (dd, 1, J = 13.2, 1.7), 3.98 (dd, 1, J = 13.2, 8.0), 3.80 (br d, 1, J = 8.0),
3.34 (q, 3, JH,F =1.8, OMe), 2.23 (ddd, 1, J = 12.8, 11.2, 10.4), 2.02 (ddd, 1, J = 12.8, 9.2, 4), 0.91-0.83 (m,
1), 0.73-0.63 (m, 1).
Partial data not including the phenyl protons for 16minor: 1H NMR  6.56 (d, 1, J = 7.6), 6.41 (dd, 1, J =
7.6, 1.2), 5.25 (dd, 1, J = 7.6, 7.2), 3.88 (dd, 1, J = 12.8, 7.6), 3.78 (br d, 1, J = 12.8), 3.77 (q, 3, JH,F = 1.8,
OMe), 3.63 (br d, 1, J = 7.6), 2.66 (ddd, 1, J = 12.8, 11.2, 10.4), 2.54-2.45 (m, 1), 2.35-2.27 (m, 1),
1.67-1.59 (m, 1).
ACKNOWLEDGEMENTS
We are grateful to the National Institutes of Health (GM-50151) for support of this work.
REFERENCES
1.
J. W. Daly, H. M. Garraffo, T. F. Spande, M. W. Decker, J. P. Sullivan, and M. Williams, Nat. Prod.
Rep., 2000, 17, 131; (b) F. I. Carroll, Biorg. Med. Chem. Lett., 2004, 14, 1889; (c) J. W. Daly, Cell.
Mol. Neurobiol., 2005, 25, 513; (d) H. M. Garraffo, T. F. Spande, and M. Williams, Heterocycles,

HETEROCYCLES, Vol. 88, No. 1, 2014
787
2009, 79, 207.
2.
3.
4.
R. W. Fitch, T. F. Spande, H. M. Garraffo, H. J. C. Yeh, and J. W. Daly, J. Nat. Prod., 2010, 73, 331.
Q. Zhou and B. B. Snider, Org. Lett., 2011, 13, 526.
(a) T. R. Hoye and M. K. Renner, J. Org. Chem., 1996, 61, 2056 and 2006, 71, 1754; (b) T. R. Hoye
and M. K. Renner, J. Org. Chem., 1996, 61, 8489; (c) T. R. Hoye, M. K. Renner, and T. J.
Vos-DiNardo, J. Org. Chem., 1997, 62, 4168.
5.
For applications to monosubstituted pyrrolidines and piperidines, see: (a) A. Jossang, A Melhaoui,
and B. Bodo, Heterocycles, 1996, 43, 755; (b) H. M. L. Davies, J. J. Matasi, L. M. Hodges, N. J. S.
Huby, C. Thornley, N. Kong, and J. H. Houser, J. Org. Chem., 1997, 62, 1095; (c) C. J. Dinsmore and
C. B. Zartman, Tetrahedron Lett., 2000, 41, 6309; (d) D. C. Beshore and C. J. Dinsmore, Tetrahedron
Lett., 2000, 41, 8735; (e) H. M. L. Davies and L. M. Hodges, J. Org, Chem., 2002, 67, 5683; (f) C.-Q.
Kang, H.-Q. Guo, X.-P. Qiu, X.-L. Bai, H.-B. Yao, and L.-X. Gao, Mag. Reson. Chem., 2006, 44, 20;
(g) P. Vidal, C. Pedregal, N. Díaz, H. Broughton, J. L. Aceña, A. Jiménez, and J. F. Espinosa, Org.
Lett., 2007, 9, 4123; (h) D. T. Tran, H. D. T. Mai, V. C. Pham, V. H. Nguyen, M. Litaudon, F. Guéritte,
Q. V. Nguyen, T. A. Tran, and V. M. Chau, Phytochem. Lett., 2013, 6, 79.
6.
For the related analysis of a 2-substituted pyrrolidine with a methoxyphenylacetamide, see: J. Gao, H.
Haas, K. Y. Wang, Z. Chen, W. Breitenstein, and S. Rajan, Mag. Reson. Chem., 2008, 46, 17.
PCMODEL 8 from Serena Software, Bloomington, IN was used for MMX calculations.
D. E. Ward and C. K. Rhee, Tetrahedron Lett., 1991, 32, 7165.
7.
8.
9.
(a) S. R. Fletcher, R. Baker, M. S. Chambers, R. H. Herbert, S. C. Hobbs, S. R. Thomas, H. M. Verrier,
A. P. Watt, and R. G. Ball, J. Org. Chem., 1994, 59, 1771; (b) A. P. Watt, H. M. Verrier, and D.
O'Connor, J. Liq. Chromatogr., 1994, 17, 1257.
10. A. A. Pandya, K. E. Panter, B. B. Snider, Y. Xiao, J. L. Yakel, Q. Zhou, and R. W. Fitch, manuscript in
preparation.